Ticks and Tick-borne Diseases 10 (2019) 868–874

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Ticks and Tick-borne Diseases

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Original article First detection of TBE virus in ticks and sero-reactivity in goats in a non- endemic region in the southern part of (Canton of ) T ⁎ Simona Casati Pagania, , Simona Frigerio Malossab, Christine Klausc, Donata Hoffmannd, Ottavio Berettaa, Nicola Bomio-Pacciorinie, Mario Lazzaroa, Giorgio Merlania, Rahel Ackermannf, Christian Beuretg a Dipartimento della sanità e della socialità, Ufficio del medico cantonale, Via Dogana 16, 6500, , Switzerland b EOC – Dipartimento di medicina di laboratorio, Servizio di microbiologia (SMIC), Via Mirasole 22a, 6500, Bellinzona, Switzerland c Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743, Jena, Germany d Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Südufer 10, 17493, Greifswald-Insel Riems, Germany e Dipartimento del territorio, Ufficio forestale 4° circondario, Via Antonio Ciseri 13, 6600, , Switzerland f National Reference Centre for Tick-Transmitted Diseases (NRZK), Labor Spiez, Ausstrasse, 3700, Spiez, Switzerland g Spiez Laboratory, Ausstrasse, 3700, Spiez, Switzerland

ARTICLE INFO ABSTRACT

Keywords: In Switzerland, tick-borne encephalitis (TBE) is a notifiable human disease with an average of 210 cases per year TBEV in the last 10 years (2008–2017). A national surveillance conducted in 2009 reported a prevalence of 0.46% for Goats tick-borne encephalitis virus (TBEV) detected in ticks, which is in accordance with the prevalences found in Ticks Europe from 0.1%–5%. The Canton of Ticino in the southern part of Switzerland, geographically separated from Ixodes ricinus the rest of the national territory by the Alps, is considered a non-endemic region, as no autochthonous clinical Switzerland cases and no TBEV presence in ticks have ever been reported. In order to understand the epidemiological si- Seroprevalence fi Sentinel host tuation in Ticino, we conducted a large study investigating the TBEV presence in eld-collected Ixodes ricinus ticks and in goat and human sera. Goats and sheep were considered as sentinel hosts showing persistence of antibodies also after 28 months in the absence of symptoms; this longevity supports the data validity to char- acterize an area with the TBEV status. The goat sera collection was composed of a total of 662 samples from 37 flocks. The total seroprevalence was 14.6%. 39 (40%) of the 97 SNT-positive samples showed an antibody titer ≥ 1:120 which indicates recent infection and consequently the probable presence of active foci among the pastures frequented by the goats belonging to 10 flocks. In total, 51 owners participated in the study and all were TBEV antibody-free. A total of 12’052 I. ricinus ticks (nymphs and adults) were collected and 1’371 pools were tested using quantitative real-time RT-PCR. Only one positive pool was reported with a prevalence of 0.35%. Metagenomic analysis revealed that the TBEV strain isolated from the ticks collected in Ticino is closely related to 2 strains coming from the Canton of (99.1% and 98.7% identity, respectively), a neighbouring region of the Canton of Ticino. These two Cantons are close together but separated by high mountains (Alps) and we hypothesize that infected ticks were transported by wild animals from Valais into the Valle in Ticino where we found positive ticks. In conclusion, our data show for the first time the presence of TBEV in ticks and the related sero- reactivity in goats, confirming the presence of TBEV in the environment of the Canton of Ticino. Further sur- veillance studies will have to be conducted to follow the persistence of TBEV in this region.

1. Introduction carry various pathogens with the potential to generate serious human and animal diseases. In Switzerland, the two most frequent tick-borne Ticks are considered the second most important vector for human human diseases are Lyme borreliosis and tick-borne encephalitis (TBE). diseases worldwide after the mosquitoes (Parola and Raoult, 2001). In The first disease caused by the bacterial species complex Borrelia Europe, the most widespread tick species is Ixodes ricinus, which may burgdorferi sensu lato is spread in Swiss forests up to an altitude of

⁎ Corresponding author at: Dipartimento della sanità e della socialità, Ufficio del medico cantonale, Via Dogana 16, 6500, Bellinzona, Switzerland. E-mail address: [email protected] (S. Casati Pagani). https://doi.org/10.1016/j.ttbdis.2019.04.006 Received 15 November 2018; Received in revised form 28 March 2019; Accepted 10 April 2019 Available online 18 April 2019 1877-959X/ © 2019 Published by Elsevier GmbH. S. Casati Pagani, et al. Ticks and Tick-borne Diseases 10 (2019) 868–874

Fig. 1. Distribution of the positive tick pool (orange star), the seropositive flocks with an antibody titer ≥ 1:120 (active foci, red dot), the seropositive flocks (past infection, orange dot) and the seronegative flocks (grey dot) in Canton of Ticino. The TBEV strains were isolated in two endemic areas in Canton of Valais (blue star) and in a pool of ticks in (orange star). The Canton of Ticino and the Canton of Valais are separated by high mountains (Alps) partly situated in . The red line shows the game ways between the two Cantons supporting the transfer of the TBEV from Valais to Ticino.

1’500 m (Medlock et al., 2013) overlapping the distribution of I. ricinus. considered as amplifier hosts play an important role in the enzootic In contrast, the tick-borne encephalitis virus (TBEV) shows an irregular transmission cycle of TBEV (Randolph et al., 1999). However, these distribution over a large geographical range with a patchy occurrence hosts develop a short viraemic phase (2-3 days) resulting in a low viral in restricted foci of limited size (Dobler et al., 2011), where the TBEV transmission potential to ticks. An essential element for virus main- circulates among tick and vertebrate populations (Charrel et al., 2004; tenance is the non-viraemic transmission between infected nymphs and Dumpis et al., 1999). non-infected larvae co-feeding on the same host without viraemia In Switzerland, natural foci are defined by registering the numbers (Randolph, 2001). Small animals develop an immune response which of autochthonous human cases and/or detection of the virus in ticks and consists in the development of persistent TBEV-specific antibodies are reported on a map published on the website of the Swiss Federal (Tonteri et al., 2016). Therefore, an alternative and complementary Office of Public Health (www.bag.admin.ch). This map indicates the way to detect TBE foci is the serological approach as demonstrated by areas where vaccination against TBEV is recommended. A national Burri who tested rodents captured at four endemic sites in Switzerland study on the prevalence of TBEV in ticks reported a mean virus pre- (Burri et al., 2012). Other animals like goats and sheep show a regular valence of 0.46% (Gäumann et al., 2010), which is in accordance with persistence of antibodies after subclinical infection. These animals live the prevalence of 0.1%–5% found in Europe (Dumpis et al., 1999; in specific areas over prolonged periods where they eventually acquire Oehme et al., 2002; Randolph, 2001). Another recent study showed the TBEV through repetitive tick bites (Klaus et al., 2012). Therefore, the absence of TBEV in questing I. ricinus ticks collected in urban and flocks are considered as sentinels for the presence of TBEV in a specific suburban areas in Switzerland (Oechslin et al., 2017). The low pre- geographic area, thus helping to better describe the epidemiological valence, the spotted distribution and the irregular circulation of TBEV situation (Klaus et al., 2010b). makes the detection of new foci very expensive and time-consuming In Switzerland, TBE is a notifiable human disease with an average of (Klaus et al., 2010a). In addition, it must be considered that areas with 210 cases per year in the last 10 years (2008–2017) with a peak of 257 TBEV-positive ticks do not always coincide with areas defined by the cases in 2017 (UFSP, 2017). The southern part of Switzerland, precisely human TBE incidence as shown in a country-wide surveillance study in the Canton of Ticino, separated from the rest of the national territory by Switzerland in 2009 with a total of 62’343 I. ricinus ticks collected in the Alps, is considered a non-endemic region, as no autochthonous 145 places (Gäumann et al., 2010). clinical cases have ever been reported. The present study is the first Many aspects remain inexplicable being confronted with a complex report of the presence of TBEV in I. ricinus ticks in this region. As the eco-epidemiological system, characterized by an intricate interplay Canton Ticino was omitted in the already mentioned first national tick between virus, ticks and tick hosts, human exposure (Kunze and ISW, surveillance study in 2009, we conducted a canton-wide study in 2014 2018), and environmental and ecological conditions. Small rodents to elucidate the prevalence of TBEV in I. ricinus ticks in this region. The

869 S. Casati Pagani, et al. Ticks and Tick-borne Diseases 10 (2019) 868–874

Table 1 TBEV-specific antibodies in goats collected in 2014, 2015 and 2016 in 6 districts of the Canton of Ticino.

Village District Collection period Total number of sera Total SNT-positive sera SNT Number of SNT samples with an AB titre ≥ 1:120* (flock) Seroprevalence (%)

Bellinzona Bellinzona 2015 20 7 35 5 Bellinzona Bellinzona 2016 4 0 0 0 Bellinzona 2014 12 0 0 0 Isone Bellinzona 2016 3 0 0 0 Medeglia Bellinzona 2015 3 0 0 0 Bellinzona 2016 2 0 0 0 Rivera Bellinzona 2015 17 0 0 0 S. Antonio Bellinzona 2016 5 0 0 0 Campello Leventina 2016 20 0 0 0 Chironico Leventina 2016 14 5 36 3 Molare Leventina 2016 22 2 9 0 Osco Leventina 2015 20 0 0 0 Rossura Leventina 2015 20 2 10 1 Berzona Locarno 2016 10 0 0 0 Verzasca Locarno 2016 20 1 5 1 Cavigliano Locarno 2016 21 0 0 0 Locarno 2015 20 11 55 8 Locarno 2015 20 6 30 3 Ronco s/ Locarno 2016 20 0 0 0 Solduno Locarno 2016 4 0 0 0 Spruga Locarno 2016 19 0 0 0 Locarno 2015 20 17 85 4 Cademario 2016 7 1 14 0 Cadro Lugano 2015 4 0 0 0 Maglio di Colla Lugano 2016 8 1 12 0 Lugano 2015 10 1 10 0 Riviera 2014 37 3 8 3 Osogna Riviera 2016 5 0 0 0 Bosco Gurin Valle Maggia 2014 34 0 0 0 Valle Maggia 2016 23 8 35 0 (1) Valle Maggia 2014 15 2 13 0 Cerentino (2) Valle Maggia 2015 35 0 0 0 Valle Maggia 2014 38 1 3 0 Maggia (1) Valle Maggia 2014 39 2 5 0 Maggia (2) Valle Maggia 2016 27 0 0 0 Valle Maggia 2014 40 20 50 8* Prato Sornico Valle Maggia 2016 24 7 29 3* Total Valle Maggia 275 40 14.5 11 Total 5 districts 387 57 14.7 28 Total all districts 662 97 14.6 39

* SNT antibody titre ≥ 1:120 shows a very recent infection. unexpected PCR-positive result for the presence of TBEV in ticks re- diseases. The flocks were randomly chosen. Of these sera, 662 from the quired a further investigation using a metagenomics and a serological years 2014–2016 were stored at -20 °C for retrospective analysis. All approach. The TBEV was isolated and characterized and the sera of samples were analyzed using the ELISA Kit Immunozym FSME IgM and goats and humans within the TBEV-positive region were analysed in IgG (Progen GmbH, Heidelberg, DE) following the protocol described order to reflect the epidemiological situation in Ticino. by Klaus et al. (2010b). Samples positive in the ELISA were confirmed using the serum neutralization test (SNT) (Klaus et al., 2010b). 2. Materials and methods 2.3. Human serum collection and serological analysis 2.1. Ticks collection and analysis In total, 69 goat owners living in Valle Maggia were invited to The ticks were collected by flagging the low vegetation between participate in the TBEV seroprevalence study authorized by the Ethics May 4 and 19, 2014 at sixty collection points within the Canton of Committees on research involving humans of the Canton Ticino (n° Ticino. The points were chosen to obtain a representative geographic 2017−00430). The farmers received a letter including information on distribution over the whole territory. The minimal collection size per the study and were asked to complete a questionnaire and to sign a collection point was set to 400 ticks within four hours of collection. Patient Informed Consent form to participate. The questionnaire pro- Collected ticks were frozen at – 80 °C until use. For each sampling site, vided information on demographics, TBE vaccination status of the local ticks were identified and separated according to species, sex, and de- population, previous trips to TBEV endemic areas in the last 5 years, velopmental stages. Five adult ticks or 10 nymphs were pooled for possible exposures to ticks and noticed bites in the last 2 years and DNA/RNA extraction and processed by real-time RT-PCR according to consumption of raw milk from goats. Sera were stored at -20 °C until the methods described by Gäumann et al. (2010). use. All samples were analyzed by ELISA and the positive samples were confirmed by SNT (Ackermann-Gäumann et al., 2018). 2.2. Serum collection from goats and analysis 2.4. Virus isolation and shotgun metagenomics analysis of TBEV Goat sera were collected by the Cantonal Veterinary Service of the Canton Ticino in the context of the national survey on epizootic To determine the whole genome of the TBEV strain TI Cevio,

870 S. Casati Pagani, et al. Ticks and Tick-borne Diseases 10 (2019) 868–874

Fig. 2. The phylogenetic analysis shows that the TBEV strain (TBEV_CH_Cevio_2014) isolated from the ticks collected in Canton of Ticino is closely related to 2 strains from the Canton of Valais (99.1% to TBEV_CH_Raron_2014 and 98.7% to TBEV_CH_Salgesch_2014). porcine kidney stable (PS) cells were inoculated with 100 μl of PCR- visualized with the web application PAVIAN (Breitwieser and Salzberg, positive tick homogenate for virus propagation in a BSL-3 facility 2016). Trimmed reads were also assembled using SPAdes version 3.12.0 (Gäumann et al., 2010). Total RNA was extracted using the RNeasy (Bankevich et al., 2012) under the “careful” and "iontorrent" parameter Mini Kit (Qiagen, Hilden, Germany) for subsequent NGS analysis ac- and a k-mer range of 21, 33, 55, 77, 99 and 127. Assembled contigs cording to the manufacturer’s instructions. The RNA was reverse tran- were back-mapped with Bowtie2 to the most related TBEV strain to scribed using the Super Script III First-Strand (Thermo Fisher Scientific, TBEV strain TI Cevio. Phylogenetic analysis was performed with Carlsbad, CA, USA) and the cDNA was quantified with the Qubit dsDNA RAxML V8.2.7 (Geneious R11, GTR+Γ substitution model with 1000 HS Assay Kit 100 on a Qubit 3.0 fluorometer (Thermo Fisher Scientific, bootstrap replicates) (Stamatakis, 2014). Carlsbad, CA, USA). Whole genome amplification was performed using fi the PicoPlex WGA kit (Takara Bio Inc.Japan) and ampli cation pro- 2.6. Statistical analysis ducts were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). The cDNA was fragmented in Micro tubes using a Covaris Statistical analysis was performed on the global set of data sampled M220 focused ultrasonicator (60 s, 20 °C, Duty Factor 20, Cycles/Burst from three collection periods: 2014, 2015 and 2016. The null hypoth- 200 and Peak Power 50) targeting peak fragments with a mean length esis of no difference among the observed frequencies of categorical of 400 bp. The fragmented cDNA was used for library preparation using variables (Total SNT-positive sera; Number of SNT samples with an AB the Ion Plus Fragment Library Kit on the AB library builder system titre ≥ 1:120) between the groups (Ticino vs. Valle Maggia) was tested fi (Thermo Fisher Scienti c, Carlsbad, CA, USA). A ratio of 0.5x AMPure with the Pearson’s Chi-square test, setting the significance level at XP (Beckmann Coulter, United States) to sample was used to purify α = 0.05. The confidence intervals on reported frequencies were cal- DNA fragment length of > 400 bd. DNA Library mean lengths and culated at a level of 95%. Data analysis was performed using R language quality were checked using High Sensitivity DNA reagents and chips on v.3.3.2. a Bio Agilent 2100 Bioanalyzer (Agilent Technologies, United States). Emulsion PCR, enrichment and chip loading were carried out using the 3. Results Ion 530 Chip kit and the Ion 510™ & 520™ & 530™ Kit-Chef on the Ion Chef system (Thermo Fisher Scientific, Carlsbad, CA, USA). The library 3.1. Ticks was sequenced on the Ion S5 system (Thermo Fisher Scientific, Carlsbad, CA, USA). A total of 12’052 I. ricinus ticks (nymphs and adults) were collected at only 43 out of 60 established points due to adverse weather condi- ’ 2.5. Bioinformatics tions. In total, 1 371 pools were tested using quantitative real-time RT- PCR. Of these, one positive pool of nymphs was reported at the site near Reads were quality checked using the Ion Torrent Suite software Cevio located in Valle Maggia resulting in a prevalence of 0.35% version 5.8.0 and trimming was performed with Trimmomatic V0.35 to (Fig. 1). a minimum DNA fragment length of 100 bp (Bolger et al., 2014). Re- ference alignment of trimmed reads to 19 TBEV genomes belonging to 3.2. Goat sera all three subtypes (European, Siberian and Far Eastern) was performed with Bowtie2 (Langmead et al., 2009). Taxonomic classification of The goat sera collection was composed of a total of 662 samples trimmed reads was performed on read level using Kraken2 version 2.0.7 from 37 different flocks, 215 sera from 2014, 189 sera from 2015 and (Wood and Salzberg, 2014). Kraken2 classification reports were 258 from 2016. Of these sera, 139 (21%) were positive according to the

871 S. Casati Pagani, et al. Ticks and Tick-borne Diseases 10 (2019) 868–874

ELISA test and 97 (97/662, 14.6%) were confirmed by SNT. A total of 3.5. Statistical analysis 19 flocks (19/37, 51%) reacted TBEV seronegative (ELISA or SNT tests; Fig. 1). Of the 97 SNT-positive samples, 39 (39/97, 40%) showed an No statistical difference was observed between the two groups antibody titer ≥ 1:120 which indicates a very recent infection, thus the (Ticino vs Valle Maggia, data not shown). The measured frequencies probable presence of active foci into the pastures frequented by the (SNT seroprevalence and the presence of active foci) were similar and goats belonging to the 10 flocks (10/37, 27%; Table 1 and Fig. 1). The indicate that the risk is not greater in one area than the other. other SNT-positive samples coming from 8 different flocks (21%) showed lower antibody titers, which indicate a past infection (last 5 4. Discussion years) (Fig. 1). In Table 1, we reported the goat seroprevalence for the Valle Maggia region (14.5%), and for the other districts of the Canton The sampling of ticks was very successful with a total of 12’004 of Ticino (14.7%; Bellinzona, Leventina, Locarno, Lugano, Riviera). The the species I. ricinus. This high number of ticks collected is essential to total seroprevalence considering all regions including the Valle Maggia obtain a current and complete epidemiological picture of TBEV in the was 14.6%. Canton of Ticino. Molecular analysis showed for the first time the presence of TBEV in one tick pool collected near the village of Cevio in 3.3. Human sera Valle Maggia. The result is surprising, since no autochthonous human TBEV infection had ever been reported in Ticino, designated so far as a In total, 51 of 69 (74%) goat owners participated in the study. None TBEV-free region. This TBEV focus shows a prevalence of 0.35%, which of them reported a vaccination against TBEV or a previous history of is below the national mean prevalence of 0.46% (Gäumann et al., TBE, 4 individuals had a previous diagnosis of Lyme borreliosis. One 2010). human serum sample was positive in the ELISA test, but could not be To better understand the virus distribution in Valle Maggia, we confirmed by SNT. A history of yellow fever vaccination could account screened goat sera for the presence of TBEV specific antibodies and for the false positive result of this sample. obtained a seroprevalence of 14.5% (Table 1). The presence of high The target population chosen seems to be suitable for the study, antibody titres (> 1:120) in goat sera in this area might indicate the since all participants (100%) had contact with ticks in 2016 and 77% presence of two active and recent foci (Menzonio and Prato Sornico, also in 2017. In addition, 42% indicated to consume raw goat's milk, Table 1). In a large serosurveillance in Germany, the authors concluded which represents an alternative – although rare – route of infection. All that positive sera indicate unexpected TBEV foci in regions currently owners were TBEV antibody free. defined as TBEV non-risk areas (Klaus et al., 2012). In a next step, the possible contact of humans with virus-infected ticks was investigated 3.4. TBEV shotgun sequencing analysis taking the goat owners living in Valle Maggia as target group. This group was chosen based on frequent contact to livestock animals, ticks 3.4.1. Virus propagation and vegetation (pastures). In this case, the human seroprevalence was TBEV propagation on PS cells was successful; Cq-values of RT-qPCR 0% pointing out once again the complex interaction between vector- analysis showed an increase of Cq 32.9 (tick homogenate) to a Cq 12.4 virus-host. within five days. A recent study performed in a non-endemic area in southeast Norway with a TBEV prevalence of 0.14% in ticks showed a very low positive seroprevalence of 0.65% in blood donors (Larsen et al., 2014). 3.4.2. Shotgun sequencing In contrast, the seroprevalence in a highly endemic region in eastern Running a 530 Chip on an Ion S5 system yielded 13'813'274 raw Poland was 19.8% in forestry workers and 32% in farmers (Cisak et al., reads with a mean read length of 215 bp. Applying a trimming of 1998). We hypothesise that the presence of TBEV in Ticino, emphasized adapters and a filtering to a minimum fragment length of 100 bp re- by a seroprevalence of 14.6% in goats - could be too recent to have sulted in 8'403'544 trimmed reads. caused human disease. Our data confirm the local circulation of TBEV in the Canton of Ticino with the presence of active foci (10), not only in 3.4.3. Reference alignment Valle Maggia, as evidenced by the high antibody titres. No statistical Bowtie2 aligned 3.54% of all trimmed reads to 19 TBEV genomes, difference was observed between the two groups where positive ticks 6'735 reads were aligned one time and 252'346 reads > 1 time. The were found, the first within the Valle Maggia and the second within highest number of reads (68'180) were aligned to TBEV strain Kubinova other districts of the Canton Ticino. (KJ922512). Alignment analysis of all trimmed reads to KJ922512 re- Goats were also used as sentinel animals in a study performed in the sulted in 314'682 aligned reads, 249'241 (2.97%) reads with a mean Canton of Valais (located in the southwest of Switzerland) to identify length of 211 bp remained after removal of duplicates. An average new risk areas for TBE with a SNT seroprevalence of 1.7% (Rieille et al., cover of 4'151 (lowest at 16 and highest at 28'278) was obtained for the 2017). It is interesting to observe that this region has a similar epide- genome of KJ922512. miological history as the Ticino: a national tick survey in 2009 identi- fied new TBE foci in Valais, known to be non-endemic (Gäumann et al., 3.4.4. De novo assembly and back-mapping 2011). In the following years, human TBE cases were reported and tick De novo assembly using SPAdes resulted in 17'545 contigs (k-mer surveys confirmed the presence of TBEV (Rieille et al., 2014). As both 127), of which 469 could be back-mapped (Bowtie2) to the reference Cantons are close to each other, although separated by the , TBEV strain Kubinova (KJ922512). we suppose that infected ticks were transported from Valais into the Valle Maggia in Ticino by wild animals. In fact, the hunting guard of 3.4.5. Phylogenetic analysis this area confirmed that wild animals cross between the two cantons The TBEV strain isolated from ticks collected in the Cevio area is (Fig. 1). In addition, phylogenetic analysis showed that the TBE strain closely related to 2 strains isolated in the Canton of Valais (99.1% to isolated in Ticino is closely related to those present in Valais (respec- TBEV_CH_Raron_2014 and 98.7% to TBEV_CH_Salgesch_2014, Fig. 2), a tively 98.7 and 99.1%). Considering that all the SNT-positive goats - neighbouring Canton of the Canton Ticino (Fig. 1). These 3 strains except two - were born in Ticino and have never left the region (per- (southern group; Fig. 2) show a close relationship with the reference sonal communication), this result led to the assumption that the origins TBEV strain Kubinova (KJ922512). However, TBEV strains from the of the TBEV virus Ticino could be in the Canton of Valais. north of Switzerland (represented by TBEV strain Spiez; Fig. 2) cluster A limitation of our study is the random assortment of the goat sera, to TBEV strain Absettarov (AF091005). which is due to the retrospective analysis of the sera available in our

872 S. Casati Pagani, et al. Ticks and Tick-borne Diseases 10 (2019) 868–874 veterinary department. Indeed all flocks were analysed only once over Breitwieser, F.P., Salzberg, S.L., 2016. Pavian: interactive analysis of metagenomics data the 3 years and smaller and bigger flocks (2–20 goats) were compared. for microbiomics and pathogen identification. bioRxiv. https://doi.org/10.1101/ 084715. Moreover, antibody detection applied does not provide any information Brockmann, S.O., Oehme, R., Buckenmaier, T., Beer, M., Jeffery-Smith, A., Spannenkrebs, on the time and place of infection. However, our data show the pre- M., Haag-Milz, S., Wagner-Wiening, C., Schlegel, C., Fritz, J., Zange, S., Bestehorn, sence of active TBEV foci (high antibody titres) through serum samples M., Lindau, A., Hoffmann, D., Tiberi, S., Mackenstedt, U., Dobler, G., 2018. A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised collected in 2014, 2015 and 2016, emphasizing the long lasting pre- goat milk and cheese in Germany, May 2016. Euro Surveill. 23. sence of active foci. Making abstraction of the temporal concept, we Burri, C., Korva, M., Bastic, V., Knap, N., Avsic-Zupanc, T., Gern, L., 2012. Serological still have the confirmation of TBEV presence in Ticino. In addition, evidence of tick-borne encephalitis virus infection in rodents captured at four sites in – TBEV specific antibodies in goats and in sheep are still detectable after Switzerland. J. Med. Entomol. 49, 436 439. Charrel, R.N., Attoui, H., Butenko, A.M., Clegg, J.C., Deubel, V., Frolova, T.V., Gould, 28 months; this longevity supports the validity to characterize an area E.A., Gritsun, T.S., Heinz, F.X., Labuda, M., Lashkevich, V.A., Loktev, V., Lundkvist, by the TBEV status (Klaus et al., 2014). A., Lvov, D.V., Mandl, C.W., Niedrig, M., Papa, A., Petrov, V.S., Plyusnin, A., An important point that emerged was the consumption of raw goat Randolph, S., Süss, J., Zlobin, V.I., de Lamballerie, X., 2004. Tick-borne virus diseases of human interest in Europe. Clin. Microbiol. Infect. 10, 1040–1055. milk by the owners (41.6%), representing an alternative route of TBEV Cisak, E., Sroka, J., Zwolinski, J., Uminski, J., 1998. Seroepidemiologic study on tick- transmission. In fact, the virus is secreted in low concentrations in goat borne encephalitis among forestry workers and farmers from the Lublin region (eastern Poland). Ann. Agric. Environ. Med. 5, 177–181. milk during the viraemic phase (Balogh et al., 2010); TBEV could be ff – Dobler, G.H., Pfe er, M., Essbauer, S., 2011. Tick-borne encephalitis: from microfocus to detected for 8 19 days in milk samples in Hungary (Balogh et al., human disease. In: Mehlhorn, H. (Ed.), Progress in Parasitology. Springer-Verlag, 2012). Although rare, a few milk-borne cases are described in the lit- Berlin Heidelberg, pp. 323–331. erature in endemic areas, for example in Slovenia (Hudopisk et al., Dumpis, U., Crook, D., Oksi, J., 1999. Tick-borne encephalitis. Clin. Infect. Dis. 28, 882–890. 2013), in Croazia (Markovinovic et al., 2016), in Hungary (Balogh Gäumann, R., Mühlemann, K., Strasser, M., Beuret, C.M., 2010. High-throughput proce- et al., 2010), in Germany (Brockmann et al., 2018) and in Slovakia dure for tick surveys of tick-borne encephalitis virus and its application in a national (Kerlik et al., 2018). In the Canton of Ticino, this transmission route is surveillance study in Switzerland. Appl. Environ. Microbiol. 76, 4241–4249. Gäumann, R., Ruzek, D., Mühlemann, K., Strasser, M., Beuret, C.M., 2011. Phylogenetic not known so far, but our data on the local goat seroprevalence impose and virulence analysis of tick-borne encephalitis virus field isolates from Switzerland. an accurate and specific information for farmers to underline the im- J. Med. Virol. 83, 853–863. portance of pasteurization processes. Hudopisk, N., Korva, M., Janet, E., Simetinger, M., Grgic-Vitek, M., Gubensek, J., Natek, In conclusion, our data show for the first time the presence of TBEV V., Kraigher, A., Strle, F., Avsic-Zupanc, T., 2013. Tick-borne encephalitis associated with consumption of raw goat milk, Slovenia, 2012. Emerg. Infect Dis. 19, 806–808. at two levels in the Canton of Ticino, once in ticks and second by the Kerlik, J., Avdicova, M., Stefkovicova, M., Tarkovska, V., Pantikova Valachova, M., sero-reactivity in goats confirming its diffusion in the environment. Molcanyi, T., Mezencev, R., 2018. Slovakia reports highest occurrence of alimentary tick-borne encephalitis in Europe: analysis of tick-borne encepahlitis outbreaks in Currently, no human TBE cases have been reported and the Canton of – fi Slovakia during 2007-2016. Travel Med. Infect. Dis. 26, 37 42. Ticino is still de ned as non-endemic area. Further epidemiological Klaus, C., Hoffmann, B., Hering, U., Mielke, B., Sachse, K., Beer, M., Süss, J., 2010a. Tick- surveillance studies have to be planned to follow more closely the borne encephalitis (TBE) virus prevalence and virus genome characterization in field- spread of TBEV in the environment. collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany. Clin. Microbiol. Infect. 16, 238–244. Klaus, C., Hoffmann, B., Moog, U., Schau, U., Beer, M., Süss, J., 2010b. Can goats be used Conflict of interest as sentinels for tick-borne encephalitis (TBE) in nonendemic areas? Experimental studies and epizootiological observations. Berl. Munch. Tierarztl. Wochenschr. 123, – fl 441 445. All of the authors declare no con icts of interest related to this ar- Klaus, C., Beer, M., Saier, R., Schau, U., Moog, U., Hoffmann, B., Diller, R., Süss, J., 2012. ticle. Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus–epidemiological studies in areas endemic and non-endemic for TBE virus in Germany. Ticks Tick. Dis. 3, 27–37. Acknowledgments Klaus, C., Ziegler, U., Kalthoff, D., Hoffmann, B., Beer, M., 2014. Tick-borne encephalitis virus (TBEV) - findings on cross reactivity and longevity of TBEV antibodies in animal We thank the Swiss Army for the support and collection of the ticks. sera. BMC Vet. Res. 10, 78. https://doi.org/10.1186/1746-6148-10-78. fi We thank the Veterinary Service of Canton Ticino for providing the Kunze, U., ISW, T., 2018. 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